In Search of High-Yielding and Single-Compound-Yielding Plants: New Sources of Pharmaceutically Important Saponins from the Primulaceae Family.

So far, only a few primrose species have been analyzed regarding their saponin composition and content. Moreover, the roots of only two of them are defined by the European Union (EU) Pharmacopoeia monograph and commercially utilized by the pharmaceutical industry. Thus, this study intended to find some new sources of main triterpene saponins from Primulae radix, namely primulasaponins I and II together with the closely related sakurasosaponin. Using isolated standards, UHPLC-ESI-HRMS served to assess over 155 Primulaceae members qualitatively and quantitatively. Nine examples of plants accumulating over 5% of primulasaponin I in their roots were found. Among them, in one case, it was found as the almost sole secondary metabolite with the concentration of 15-20% (Primula grandis L.). A reasonable content of primulasaponin II was found to be typical for Primula vulgaris Huds. and P. megaseifolia Boiss. & Bal. The sakurasosaponin level was found in seven species to exceed 5%. The finding of new, single and rich sources of the abovementioned biomolecules among species that were never analyzed phytochemically is important for future research and economic benefit. The chemotaxonomic significance of the occurrence of these three saponins in Primulaceae is discussed.

Chloroplast Genomes and Comparative Analyses among Thirteen Taxa within Myrsinaceae s.str. Clade (Myrsinoideae, Primulaceae).

The Myrsinaceae s.str. clade is a tropical woody representative in Myrsinoideae of Primulaceae and has ca. 1300 species. The generic limits and alignments of this clade are unclear due to the limited number of genetic markers and/or taxon samplings in previous studies. Here, the chloroplast (cp) genomes of 13 taxa within the Myrsinaceae s.str. clade are sequenced and characterized. These cp genomes are typical quadripartite circle molecules and are highly conserved in size and gene content. Three pseudogenes are identified, of which ycf15 is totally absent from five taxa. Noncoding and large single copy region (LSC) exhibit higher levels of nucleotide diversity (Pi) than other regions. A total of ten hotspot fragments and 796 chloroplast simple sequence repeats (SSR) loci are found across all cp genomes. The results of phylogenetic analysis support the notion that the monophyletic Myrsinaceae s.str. clade has two subclades. Non-synonymous substitution rates (dN) are higher in housekeeping (HK) genes than photosynthetic (PS) genes, but both groups have a nearly identical synonymous substitution rate (dS). The results indicate that the PS genes are under stronger functional constraints compared with the HK genes. Overall, the study provides hypervariable molecular markers for phylogenetic reconstruction and contributes to a better understanding of plastid gene evolution in Myrsinaceae s.str. clade.

Molecular thresholds of ITS2 and their implications for molecular evolution and species identification in seed plants.

Although molecular data have revealed huge amounts of plant diversity, interpreting genetic diversity into entities corresponding to species is still challenging. Taxonomic ranking based on genetic distance has been used extensively, but the results have been open to dispute, while the application of the strategy to plants has been restricted to a small number of cases. Here, levels of internal transcribed spacer 2 (ITS2) sequence variation were examined from 17,203 sequences, representing 5,439 species in 113 genera of seed plants, to ascertain the association between species status and their molecular divergence. Our results showed that, although the average genetic distances of sister species (AGDS) varied among angiosperms, the mean value was 3.98% and seemed not to be influenced by higher-level hierarchical classification or life history. AGDS was also stable within the major lineages of the gymnosperms but at approximately half the value of angiosperms, except for the Gnetidae, where the AGDS almost equaled that of angiosperms. We found that these AGDS discrepancies, associated with the rates of molecular evolution, cannot simply be attributed to generation-time differences, and highlight the complex life histories of plants. Our results provide general ITS2 thresholds in seed plants, and suggest their use in species identification.

[Analysis of varieties and standards of Primulaceae medicinal plants used in Tibetan medicine].

In this paper, the varieties and origin of Primulaceae plants that used in Tibetan medicine were analyzed. The results showed that there were 3 genera and 44 species (including the varieties) of Primulaceae plants were recorded in the relevant literatures. Among them, 17 varieties were recorded in Tibetan names, 24 varieties were recorded in Chinese names and 1 variety was used in both of them. In current quality criteria of standards at all levels in China country, 6 varieties were recorded in Tibetan names and 6 original plants were involved, which were 35% and 14% of them respectively. Seventeen varieties were recorded in Chinese name and 7 original plants were involved, which were 30% and 16% of them respectively. In Tibetan medicine standards and literatures, there were big differences between Tibetan names and Chinese names which were translated from Tibetan names and its original plants. There were only regulations of morphological identification and microscopic authentication, so the standards were very inadequate. Therefore, through literatures research, resources and current situation investigations, combining the research and specification of the name and original of Tibetan medicine, the level of normalization and standardization could be enhanced, the stable and controllable safety and utility in clinical medication could be ensured to promote advancement of industry technology Tibetan medicine.

Evolution, ecology and systematics of Soldanella (Primulaceae) in the southern Apennines (Italy).

BACKGROUND: The populations of Soldanella (Primulaceae) of the southern Apennines (Italy) are unique within the genus for their distribution and ecology. Their highly fragmented distribution range, with three main metapopulations on some of the highest mountains (Gelbison, Sila and Aspromonte massifs) of the area, poses intriguing questions about their evolutionary history and biogeography, and about the possibility of local endemisms. AIMS AND METHODS: In order to clarify the phylogeny and biogeography of the three metapopulations of Soldanella in the southern Apennines, attributed to S. calabrella to date, and to identify possible local endemisms, a comparative approach based on the study of molecular, morphological and ecological characteristics of the populations was employed. Specifically, one nuclear (total ITS) and two plastid (rbcL and trnL) markers were used for the phylogenetic analyses, performed through both maximum likelihood and Bayesian techniques. Among the morphological features, the glandular hair and leaf biometric traits were analysed, and the environment in which the populations grew was characterised for altitude, forest canopy composition and soil pH, C, N and organic matter. RESULTS AND CONCLUSIONS: Our findings demonstrate that the lineage of Soldanella of southern Italy diverged from the Carpathians lineage during the Middle Pleistocene, and underwent an evolutionary radiation during the Late Pleistocene. The populations of the Sila and Aspromonte massifs diverged from the populations of the Gelbison massif around 380000 years ago and are probably undergoing a progressive differentiation due to their isolation. The populations on the Gelbison massif, moreover, have different morphological features from those of the Sila and Aspromonte massifs and a different ecological niche. The molecular, morphological and ecological data clearly demonstrate that the metapopulation of Soldanella on the Gelbison massif belongs to a new taxonomic unit at the species level, which we name Soldanella sacra A. & L. Bellino from the name of the massif on which it was discovered, the "Holy Mountain".

Automation and Evaluation of the SOWH Test with SOWHAT.

The Swofford-Olsen-Waddell-Hillis (SOWH) test evaluates statistical support for incongruent phylogenetic topologies. It is commonly applied to determine if the maximum likelihood tree in a phylogenetic analysis is significantly different than an alternative hypothesis. The SOWH test compares the observed difference in log-likelihood between two topologies to a null distribution of differences in log-likelihood generated by parametric resampling. The test is a well-established phylogenetic method for topology testing, but it is sensitive to model misspecification, it is computationally burdensome to perform, and its implementation requires the investigator to make several decisions that each have the potential to affect the outcome of the test. We analyzed the effects of multiple factors using seven data sets to which the SOWH test was previously applied. These factors include a number of sample replicates, likelihood software, the introduction of gaps to simulated data, the use of distinct models of evolution for data simulation and likelihood inference, and a suggested test correction wherein an unresolved "zero-constrained" tree is used to simulate sequence data. To facilitate these analyses and future applications of the SOWH test, we wrote SOWHAT, a program that automates the SOWH test. We find that inadequate bootstrap sampling can change the outcome of the SOWH test. The results also show that using a zero-constrained tree for data simulation can result in a wider null distribution and higher p-values, but does not change the outcome of the SOWH test for most of the data sets tested here. These results will help others implement and evaluate the SOWH test and allow us to provide recommendations for future applications of the SOWH test. SOWHAT is available for download from https://github.com/josephryan/SOWHAT.

Lysimachia huangsangensis (Primulaceae), a New Species from Hunan, China.

A new species, Lysimachia huangsangensis (Primulaceae), from Hunan, China is described and illustrated. The new species is closely related to L. carinata because of the crested calyx, but differs in the leaf blades that are ovate to elliptic and (3-)4.5-9 × 2-3.4 cm, 2-5-flowered racemes, and the calyx lobes that are ovate-lanceolate and 5-6 × 3-4 mm. The systematic placement and conservation status are also discussed.

Fatty acid composition, antioxidant and antibacterial properties of the microwave aqueous extract of three varieties of Labisia pumila Benth.

BACKGROUND: The present study was conducted in order to evaluate the fatty acid profile, anti-oxidant and anti-bacterial activities from the microwave aqueous extract of the leaves of three different varieties of Labisia pumila Benth. RESULTS: The chemical analysis of the extract showed that fatty acids (palmitic, palmitoleic, stearic, oleic, linoleic and α-linolenic) acid as the main components in three varieties of L. pumila leaves. Furthermore, the obtained results of the anti-oxidant revealed that L. pumila var. alata contained higher anti-oxidative activities compared to var. pumila and var. lanceolata. However, these values were lower than the tested anti-oxidant standards. On the other hand, the aqueous leaf extracts in all three varieties of L. pumila were also found to inhibit a variable degree of antibacterial activities against eight bacteria (four Gram-positive and four Gram-negative bacteria). CONCLUSIONS: In this study, it was observed the leaves of three varieties of L. pumila exhibited variable patterns of fatty acids and the microwave aqueous extraction possess anti-oxidant and anti-bacterial activities.

Fatty acid composition, antioxidant and antibacterial properties of the microwave aqueous extract of three varieties of Labisia pumila Benth

BACKGROUND: The present study was conducted in order to evaluate the fatty acid profile, anti-oxidant and anti-bacterial activities from the microwave aqueous extract of the leaves of three different varieties of Labisia pumila Benth. RESULTS: The chemical analysis of the extract showed that fatty acids (palmitic, palmitoleic, stearic, oleic, linoleic and α-linolenic) acid as the main components in three varieties of L. pumila leaves. Furthermore, the obtained results of the anti-oxidant revealed that L. pumila var. alata contained higher anti-oxidative activities compared to var. pumila and var. lanceolata. However, these values were lower than the tested anti-oxidant standards. On the other hand, the aqueous leaf extracts in all three varieties of L. pumila were also found to inhibit a variable degree of antibacterial activities against eight bacteria (four Gram-positive and four Gram-negative bacteria). CONCLUSIONS: In this study, it was observed the leaves of three varieties of L. pumila exhibited variable patterns of fatty acids and the microwave aqueous extraction possess anti-oxidant and anti-bacterial activities.

Volatiles of Lysimachia paridiformis Var. Stenophylla, Lysimachia fortumei and Lysimachia chikungensis by HS-SPME-GC-MS.

BACKGROUND: Lysimachia paridiformis Var. Stenophylla mainly contain flavonoid constituents. Flavonoids and benzoquinones are the main compounds in L. fortumei Maxim. The objective of this paper was to study the volatile compounds of leaves in L. paridiformis Var. Stenophylla, L. fortumei and L. chikungensis for the first time. MATERIALS AND METHODS: Volatiles were extracted by the manual solid phase micro-extraction (SPME). The volatile constituents were analyzed by an Agilent 6890 N gas chromatograph equipped and coupled with a 5975B mass selective detector spectrometer. RESULTS: Twenty-nine compounds were identified in the leaves of L. paridiformis var. Stenophylla, accounting for 89.17% of the total volatile fraction. The main constituents were ethanol (13.58%), and ß-ionone (8.05%). linalool and ß-ionone were the main aroma constituents in L. paridiformis var. Stenophylla. Twenty-one compounds were identified in the leaves of L. fortumei, accounting for 94.72% of the total volatile fraction. The main constituents were tricosane (14.72%), docosane (11.02%), tetracosane (10.77%) and pentacosane (9.81%). Thirty-two compounds were identified in the leaves of L. chikungensis, accounting for 88.58% of the total volatile fraction. Typical compounds detected in L. chikungensis were cis-3-hexenyl pentanoate (13.33%), followed by ethanol (12.13%), ethyl palmitate (7.78%), and heneicosane (5.38%). CONCLUSION: The results showed that the main composition types were similar in the three plants, but the content was different, which indicated that the similar composition types provided the same medical effect for three plants.

[Optimization for ISSR-PCR system of traditional Chinese medicine Lysimachia christinae by orthogonal design].

In order to establish the stable andreliable ISSR-PCR System of Lysimachia christinae, L16 (4(5)) orthogonal design, which based on 7 levels of single factor experiment, were used in this study. The variance analysis was carried out by SPSS 19.0, and 5 main factors affecting the reaction system were optimized in 4 levels. The best annealing temperature was selected by the optimized reaction system. And the stability and reliability of this system was tested by 23 samples from different origins. The results showed that the five factors (DNA template, primer, dNTP, Mg2+ and Taq enzyme) were the most impacts on the amplified results of ISSR-PCR of L. christinae. The order of the influence was: primer > Taq enzyme > DNA template > Mg2+ > dNTP. The optimal system, which was determined by multiple comparison on different levels of each factor, was total volume of 25 microL, including DNA template 60 ng, primer 0.3 micromol x L(-1), dNTP 0.2 mmol x L(-1), Mg2+ 1.8 mmol x L(-1), Taq enzyme 1.25 U. The optimal system was stable and reliable tested by 23 samples from different origins. This study lays the foundation for genetic diversity analysis, fine varieties selection and molecular identification of L. christinae, and provides reference for optimization on ISSR-PCR system of other speciesin future.

Antifungal, anti-inflammatory and cytotoxicity activities of three varieties of labisia pumila benth: from microwave obtained extracts.

BACKGROUND: Labisia pumila, locally known as Kacip Fatimah, is a forest-floor plant that has tremendous potential in the herbal industry. It is one of the five herbal plants identified by the government as one of the national key economic areas to be developed for commercial purposes. There are three varieties of L. pumila namely, L. pumila var. pumila, L. pumila var. alata and L. pumila var. lanceolata and each has its own use. METHODS: The leaves and roots of the three varieties of L. pumila Benth. were extracted using microwave assisted extraction (MAE). Antifungal activity of all plant extracts were characterized against Fusarium sp., Candida sp. and Mucor using the agar diffusion disc. Anti-inflammatory assays were performed using NO production by macrophage RAW 264.7 cell lines induced by LPS/IFN-g and cytotoxic activity was determined using several cancer cell lines and one normal cell line. RESULTS: The overall result demonstrated that leaf and root extracts of all three varieties of L. pumila exhibited moderate to appreciable antifungal activity against Fusarium sp., Candida sp. and Mucor compared to streptomycin used as positive control. Leaf and root extracts of all varieties significantly decreased NO release. However, the root extracts showed higher activity compared to the leaf extracts. Cytotoxic activity against MCF-7, MDA-MB-231 and Chang cell lines were observed with all extracts. CONCLUSIONS: These findings suggest the potential use of L. pumila Benth. as a natural medicine and indicated the possible application of this medicinal plant such anti inflammatory activity and cytotoxic agents.

[Study on HPLC fingerprint of Herba Lysimachiae].

OBJECTIVE: To establish the chromatographic fingerprint for Herba Lysimachiae by RP-HPLC. METHODS: The sample solutions were prepared by extracting material with boiling water,followed by extracting with BuOH from the aqueous extract and analyzed by RP-HPLC with a Phenomenex C18 column (4.6 mm x 150 mm, 5 microm), gradiently eluted with A (acetonitrile containing 0.5% of acetic acid)-B (water containing 0.5% of acetic acid) at 30 degrees C, and monitored with UV detector at 255 nm. The flow rate was 1.0 mL/min, and sample volume injected was 20 microL. The chromatograms of different batches of Herba Lysimachiae were compared by the software of Similarity Evaluation System for Chromatographic Fingerprint of TCM (Version 2004A). RESULTS: HPLC Fingerprint of Herba Lysimachiae was established with Rutin as the reference compound. 13 common peaks were selected as the fingerprint peaks of Herba Lysimachiae. CONCLUSION: The established HPLC fingerprint of Herba Lysimachiae has desirable precision, reproducibility, and can be applied to routine quality control of Herba Lysimachiae.

Floral and vegetative cues in oil-secreting and non-oil-secreting Lysimachia species.

BACKGROUND AND AIMS: Unrelated plants pollinated by the same group or guild of animals typically evolve similar floral cues due to pollinator-mediated selection. Related plant species, however, may possess similar cues either as a result of pollinator-mediated selection or as a result of sharing a common ancestor that possessed the same cues or traits. In this study, visual and olfactory floral cues in Lysimachia species exhibiting different pollination strategies were analysed and compared, and the importance of pollinators and phylogeny on the evolution of these floral cues was determined. For comparison, cues of vegetative material were examined where pollinator selection would not be expected. METHODS: Floral and vegetative scents and colours in floral oil- and non-floral oil-secreting Lysimachia species were studied by chemical and spectrophotometric analyses, respectively, compared between oil- and non-oil-secreting species, and analysed by phylogenetically controlled methods. KEY RESULTS: Vegetative and floral scent was species specific, and variability in floral but not vegetative scent was lower in oil compared with non-oil species. Overall, oil species did not differ in their floral or vegetative scent from non-oil species. However, a correlation was found between oil secretion and six floral scent constituents specific to oil species, whereas the presence of four other floral compounds can be explained by phylogeny. Four of the five analysed oil species had bee-green flowers and the pattern of occurrence of this colour correlated with oil secretion. Non-oil species had different floral colours. The colour of leaves was similar among all species studied. CONCLUSIONS: Evidence was found for correlated evolution between secretion of floral oils and floral but not vegetative visual and olfactory cues. The cues correlating with oil secretion were probably selected by Macropis bees, the specialized pollinators of oil-secreting Lysimachia species, and may have evolved in order to attract these bees.

Reconstructing the origins of high-alpine niches and cushion life form in the genus Androsace S.L. (Primulaceae).

Relatively, few species have been able to colonize extremely cold alpine environments. We investigate the role played by the cushion life form in the evolution of climatic niches in the plant genus Androsace s.l., which spreads across the mountain ranges of the Northern Hemisphere. Using robust methods that account for phylogenetic uncertainty, intraspecific variability of climatic requirements and different life-history evolution scenarios, we show that climatic niches of Androsace s.l. exhibit low phylogenetic signal and that they evolved relatively recently and punctually. Models of niche evolution fitted onto phylogenies show that the cushion life form has been a key innovation providing the opportunity to occupy extremely cold environments, thus contributing to rapid climatic niche diversification in the genus Androsace s.l. We then propose a plausible scenario for the adaptation of plants to alpine habitats.

Testing DNA barcoding in closely related groups of Lysimachia L. (Myrsinaceae).

It has been suggested that rbcL and matK are the core barcodes in plants, but they are not powerful enough to distinguish between closely related plant groups. Additional barcodes need to be evaluated to improve the level of discrimination between plant species. Because of their well-studied taxonomy and extreme diversity, we used Chinese Lysimachia (Myrsinaceae) species to test the performance of core barcodes (rbcL and matK) and two additional candidate barcodes (trnH-psbA and the nuclear ribosomal ITS); 97 accessions from four subgenus representing 34 putative Lysimachia species were included in this study. And many closely related species pairs in subgen. Lysimachia were covered to detect their discriminatory power. The inefficiency of rbcL and matK alone or combined in closely related plant groups was validated in this study. TrnH-psbA combined with rbcL + matK did not yet perform well in Lysimachia groups. In contrast, ITS, alone or combined with rbcL and/or matK, revealed high resolving ability in Lysimachia. We support ITS as a supplementary barcode on the basis of core barcode rbcL and matK. Besides, this study also illustrates several mistakes or underlying evolutionary events in Lysimachia detected by DNA barcoding.

Evidence of three parallel evolutions of leaf dwarfism and phytogeography in Lysimachia sect. Nummularia in Japan and Taiwan.

Phylogenetic analyses were performed using nrITS variations of eight species including one variety of the genus Lysimachia sect. Nummularia in Japan and Taiwan, with another 34 Lysimachia species and Glaux maritime obtained from a DNA database. Among the four dwarf-leaf taxa in Japan and Taiwan, L. japonica var. minutissima of Japan and two Taiwanese species were placed into two different clades; and L. liukiuensis of Japan, was positioned outside of these two clades. These results suggest that three independent dwarfism events generated the four dwarf-leaf taxa; and that there were likely multiple migration events from China to Japan and Taiwan.

Bayesian hypothesis testing supports long-distance Pleistocene migrations in a European high mountain plant (Androsace vitaliana, Primulaceae).

Colonization of the south-western European mountain ranges is suggested to have predominantly progressed from the Iberian Peninsula eastwards, but this hypothesis has never been tested in a statistical framework. Here, we test this hypothesis using Androsace vitaliana, a high elevation species with eight mostly allopatric subspecies, which is widely but disjunctly distributed across all major south-western European mountain ranges. To this end, we use plastid and nuclear sequence data as well as fingerprint (amplified fragment length polymorphisms) data and employ Bayesian methods, which allow co-estimation of genealogy and divergence times using explicit demographic models, as well as hypothesis testing via Bayes factors. Irrespective of the ambiguity concerning where A. vitaliana started to diversify -- both the Alps and the mountain ranges of the Iberian Peninsula outside the Pyrenees were possible -- colonization routes were not simply unidirectional, but involved Pleistocene connections between the Alps and mountain ranges of the Iberian Peninsula bypassing the interjacent Pyrenees via long-distance dispersal. In contrast, the species' post-glacial history is shaped by regional gene pool homogenization resulting in the genetic pattern showing good congruence with geographical proximity in agreement with a vicariance model, but only partly supporting current taxonomy.

Reciprocal Pleistocene origin and postglacial range formation of an allopolyploid and its sympatric ancestors (Androsace adfinis group, Primulaceae).

The biogeographic history of polyploids and their lower-ploid ancestors is an important feature to achieve a better understanding of polyploid evolution. This is exemplified here using the ecologically congruent members of the Androsace adfinis group (Primulaceae) endemic to the southwestern European Alps. Employing relative genome size, AFLP fingerprint and chloroplast sequence haplotype data, we show that Androsace brigantiaca is a recent (probably no more than 0.2 million years) allopolyploid derivative of the geographically close A. adfinis and A. puberula, which formed reciprocally in a comparatively restricted area in the southern Southwestern Alps. Bayesian admixture analysis--also of artificial additive AFLP profiles--shows that the nuclear genome of A. brigantiaca is significantly biased towards the puberula-genome irrespective of maternal parentage. Nevertheless, there is no evidence for genetic interaction (hybridization, introgression) of A. brigantiaca with either of its ancestors, including the widely sympatric A. puberula. Sympatry might be facilitated by ecological displacement on a local scale or might be a transitory phase on the way to competitive replacement via, for instance, polyploid superiority.

Fine- and landscape-scale spatial genetic structure of cushion rockjasmine, Androsace tapete (Primulaceae), across southern Qinghai-Tibetan Plateau.

The cushion rockjasmine, Androsace tapete (Primulaceae), is among the angiosperms with highest altitudal distribution in the world. Cushion rockjasmine is a prominent pioneer species in alpine deserts and alpine flowstone slope habitats up to 5,300 m on Qinghai-Tibetan Plateau. In this study, we use inter simple sequence repeat (ISSR) markers to investigate the spatial genetic structure of A. tapete at both fine-scale and landscape-scale, with emphasis on testing the hypothesis that the low-altitude valley of the Brahmaputra River, running from west to east across Qinghai-Tibetan Plateau, has significant effects on the spatial population structure of A. tapete. A total of 235 individuals were collected from five populations in disjunct ridges (i.e. two populations located in the north, and three in the south of the Brahmaputra River), including 158 individuals that were spatial explicitly sampled from a 30 m x 90 m plot. At fine scale, spatial autocorrelation analysis indicates a significant genetic structure within a short distance (less than 10 m), which is probably due to limited gene dispersal via pollen and/or seeds. At landscape scale, however, AMOVA suggests that most of the total genetic variation (85%) is among individuals within populations; and the Brahmaputra River plays a weak role in shaping the spatial population structure of A. tapete. In addition, the results of PCA and STRUCTURE assignment show significant genetic associations between the populations across the Brahmaputra River. The historical gene exchanges and slow genetic drift may be responsible for the lack of deep genetic differentiation among topographically separated populations in A. tapete.